1.1 Field of the Invention
The present invention relates generally to the fields of immunology, cancer therapy, molecular biology and cell biology. The present invention relates in particular to compositions and methods for use of insect cells containing non-surface expressed proteins or peptides, encoded by baculovirus expression vectors. Such compositions and methods may be of therapeutic use in the treatment of disease states, such as cancer.
1.2 Description of Related Art
Most progressively growing neoplasms do not provoke immunological responses sufficient to control the growth of malignant cells, despite the fact that tumor cells express antigens which are recognizable as foreign by the immune system of the patient (Sibille et al., 1990).
Tumor-associated antigens (TAAs) capable of being recognized by the cellular immune system (T-cells) have been identified. These antigens (also referred to as tumor associated or T-cell epitopes) include oncogene products activated by mutation and rearrangement (e.g., position 12 mutation in p21ras; P210 product of bcr/abl rearrangement); mutated tumor-suppressor gene products (e.g., p53); reactivated embryonic gene products not expressed in adult tissues (e.g., P91A found in the P815 mastocytoma); MAGE 1 (found in melanomas and human breast tumors); tissue specific self-antigens expressed by tumors (e.g., tyrosinase); and a variety of others (Pardoll, 1993). Most tumor cell populations express certain common TAAs, but are heterogeneous with respect to the spectrum of TAAs that they express. Despite the array of tumor-associated T-cell epitopes expressed in tumors, tumor cells remain poorly immunogenic.
An approach to genetic engineering of tumor cells is the use of viral expression vectors to infect tumor cells. Poxvirus technology has been utilized to elicit immunological responses to TAAs in animal models of experimentally-induced tumors. The gene encoding carcinoembryonic antigen (CEA) was isolated from human colon tumor cells and inserted into the vaccinia virus genome (Kaufman et al., 1991). Inoculation of the vaccinia-based CEA recombinant elicited CEA-specific antibodies and an antitumor effect in a mouse model (Id.). The human melanoma TAA, p97, has also been inserted into vaccinia virus and shown to protect mice from tumor transplants (Hu et al., 1988; Estin et al., 1988). Bernards et al. (1987) constructed a vaccinia recombinant that expressed the extracellular domain of the neu-encoded p185 glycoprotein. Mice immunized with this recombinant virus developed a strong humoral response against the neu gene product and were protected against subsequent tumor challenge.
Killing of tumor cells by the immune system is mediated by cytotoxic T-lymphocytes (CTLs). However, the recognition of tumor-associated antigens is restricted by class 1 determinants specified by the major histocompatibility complex (DeGiovanni et al., 1991; Porgador et al., 1989; Kim et al., 1994). Suppression or failure to express MHC class I antigens is one of several documented mechanisms which enable tumor cells to escape T-cell mediated host immunity (Elliott et al., 1990; Tanaka et al., 1988).
Attempts have been made to use cytokines to augment the immune response to tumor-associated antigens. The goal of this strategy is to alter the local immunological environment of the tumor cell to enhance the presentation of T-cell epitopes or the activation of tumor-specific T-lymphocytes (Pardoll, 1993). Various cytokine genes have been introduced into tumor cells. Immunization with neoplastic cells modified to secrete IL-2 (Porgador et al., 1993a; Karp et al., 1993), IFN-xcex1 (Porgador et al., 1993b) or GM-CSF (Dranoff et al., 1993), among others (Pardoll et al., 1992; Rosenberg et al., 1992), resulted in the generation of CTLs with cytotoxic activity towards both the cytokine-secreting and non-secreting tumor cells. Experimental animals and a small number of patients with established neoplasms treated with the cytokine-secreting cells survived for prolonged periods, although in most instances tumor growth eventually recurred (Id.).
Recombinant vaccinia viruses also have been used to express cytokine genes (Ruby et al., 1992). Expression of certain cytokines (IL-2, IFN-xcex1) led to self-limiting vaccinia virus infection in mice and, in essence, acted to attenuate the virus. Expression of other cytokines (i.e. IL-5, IL-6) were found to modulate the immune response to co-expressed extrinsic immunogens (Review by Ruby el al., 1992).
Although promising, these observations have not yet resulted in a clinically effective method of eliminating or substantially reducing tumor burden in individuals with cancer. In addition to being expensive, direct in vivo administration of purified cytokines may result in toxic side-effects. Genetically engineering tumor cells to express cytokines in vitro, with subsequent reintroduction into the patient, is difficult, time-consuming, expensive and of unproven clinical efficacy. Gene therapy with human infective viruses engineered to express cytokines has not yet been successfully implemented at the clinical level. One difficulty with this approach is the possible activation of replication-defective viruses by in vivo recombination with naturally occurring human viruses.
A potential solution to this problem involves using baculovirus that has been genetically engineered to express therapeutic proteins. Naturally occurring insect baculovirus infects only arthropods. The host range of insect baculoviruses has been extensively studied and no evidence of infection or pathogenic responses has been identified in non-host insects, plants, vertebrates or humans (Groner, 1986). This feature may make baculovirus an ideal agent to be modified and used for the delivery of drugs, genes, or therapeutics.
The present invention addresses deficiencies in the art by disclosing compositions and methods for use of insect cells containing an isolated nucleic acid segment encoding a selected non-surface expressed protein or peptide, for example, a therapeutic protein. A xe2x80x9cnon-surface expressed protein or peptidexe2x80x9d is defined herein as an expressed protein or peptide that is not localized to the cell membrane of the insect cell. In this sense, such proteins or peptides may potentially be secreted into the extracellular environment. Alternatively, the non-surface expressed protein or peptide may be intracellular within the insect cell.
In certain embodiments, the isolated nucleic acid segment is contained within a baculovirus expression vector. The construction of recombinant baculovirus vectors may be accomplished by techniques well known in the art.
In one aspect of the present invention, the non-surface expressed protein or peptide is a cytokine. It is contemplated that almost any cytokine could be used in the practice of the present invention. Classes of cytokines contemplated within the scope of the present invention include interferons, interleukins, tumor necrosis factors and colony stimulating factors. Examples of specific cytokines of potential use in the present invention include interleukin 1 (IL-1), IL-2, IL-5, IL-10, IL-11, IL-12, IL-18, interferon-xcex3 (IF-xcex3), IF-xcex1, IF-xcex2, tumor necrosis factors-xcex1 (TNF-xcex1), and GM-CSF (granulocyte macrophage colony stimulating factor). Such examples are representative only and are not intended to exclude other cytokines known in the art. In a particular embodiment, the cytokine is xcex2-interferon or GM-CSF.
The skilled artisan will realize that the term xe2x80x9cprotein or peptidexe2x80x9d encompasses proteins or peptides with the naturally occurring amino acid sequences of identified proteins or peptides, as well as minor sequence variants of such proteins or peptides. These may, for instance, be minor sequence variants of the polypeptide which arise due to natural variation within the population or they may be homologues found in other species. They also may be sequences which do not occur naturally but which are sufficiently similar that they function similarly and/or elicit an immune response that cross-reacts with natural forms of the polypeptide. Sequence variants can be prepared by standard methods of site-directed mutagenesis or peptide synthesis. Amino acid sequence variants of the polypeptide can be substitutional, insertional or deletion variants. Deletion variants lack one or more residues of the native protein which are not essential for function or immunogenic activity. A common type of deletion variant is one lacking secretory signal sequences or signal sequences directing a protein to bind to a particular part of a cell.
Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide such as stability against proteolytic cleavage. Substitutions preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine: glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamate; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
Insertional variants include hybrid proteins containing sequences from other proteins and polypeptides which are homologues of the polypeptide. For example, an insertional variant could include portions of the amino acid sequence of the polypeptide from one species, together with portions of the homologous polypeptide from another species. Other insertional variants can include those in which additional amino acids are introduced within the coding sequence of the polypeptide. These typically are introduced, for example, to disrupt a protease cleavage site.
In one aspect, the invention is directed to a method of providing a therapeutic protein to a mammal, comprising preparing a composition comprising insect cells containing an isolated nucleic acid segment encoding a selected non-surface expressed protein or peptide and administering the composition to a mammal. In a preferred embodiment, the mammal is a human subject. In certain embodiments, the insect cells are lyophilized or subjected to freeze-thaw cycles prior to administration. It is contemplated within the scope of the invention that administration of the composition may be accomplished by essentially any route of administration, such as intramuscular, subcutaneous, intraperitoneal, intravascular or intraarterial. For an appropriate composition, administration may occur by oral, nasal, buccal, rectal, vaginal or topical routes.
In a particular embodiment, the composition comprising insect cells may be administered by direct intratumoral injection to a mammal with cancer. The resulting activation of the host immune system, targeted to one or more TAAs, is of utility in reducing or eliminating tumor burden in the subject. This surprising result is of great significance for the clinical treatment of human cancer. Depending upon the specific cytokine selected, other mechanisms of therapeutic treatment are contemplated within the scope of the present invention. For example, xcex2-interferon may interfere with tumor growth by inhibiting angiogenesis. (See Fabra et al., 1992; Ghoji et al., 1994a, 1994b; Singh et al., 1995, 1996a, 1996b; Singh and Fidler, 1997; Dinney et al., 1998, each of which is incorporated herein by reference). In certain embodiments, the administration of compositions comprising insect cells may occur as an adjunct to or in combination with more traditional tumor therapies, such as chemotherapy, radiotherapy or immunotherapy.
The present invention further discloses the unexpected result that subjects whose tumor burdens have been eliminated are resistant to further challenge with cancers expressing the same TAA(s). Yet another surprising aspect of the present invention is that direct tumoral injection of insect cells alone may result in at least a partial suppression of tumor growth. The possibility of an additive or synergistic effect upon the host immune system of presenting both insect cells and expressed therapeutic proteins is contemplated within the scope of the present invention.
The skilled artisan will realize that methods of therapeutic treatment of cancer and methods of immunization of a subject against recurrence of tumors exhibiting the same TAA(s) are contemplated within the scope of the present invention. It further is contemplated within the scope of the present invention that insect cells expressing therapeutic proteins may be used as universal adjuvants in boosting immune system response in various other disease states, such as AIDS or influenza. The skilled artisan will realize that the term xe2x80x9cinsect cellsxe2x80x9d includes intact insect cells as well as insect cells that have been lyophilized or subjected to freeze-thaw cycles.
One aspect of the present invention concerns compositions comprising insect cells containing isolated nucleic acid segments encoding a selected non-surface expressed protein or peptide of interest. A variety of different types of insect cells are considered within the scope of the present invention, including cells from Autographa Californica Bombyx mori, Spodoptera frugiperda, Choristoneura fumiferana, Heliothis virescens, Heliothis zea, Orgyia pseudotsugata, Lymantira dispar, Plutelia xylostella, Malacostoma disstria, Trichoplusia ni, Pieris rapae, Mamestra configurata and Hyalophora cecropia. In a particular embodiment, the insect cell is a Spodoptera frugiperda cell or a Trichoplusia ni cell. In certain embodiments, such cells may include Sf9 cells or H5 cells (High Five(trademark), Invitrogen, Sorrento, Calif.). In certain embodiments, the isolated nuclcic acid segment is incorporated into a baculovirus expression vector. It is contemplated within the scope of the present invention that the baculovirus may be any baculovirus that can be engineered to express an isolated nucleic acid segment encoding a selected non-surface expressed therapeutic protein or peptide. Representative baculoviruses include AcMNPV (Autographa californica multinucleocapsid nuclear polyhedrosis virus). BmNPV (Bombyx mori nucleopolyhedrosis virus) and pBlueBac (Invitrogen, Sorrento, Calif.). See U.S. Pat. No. 4,215,051 (incorporated herein by reference).
Another embodiment of the present invention encompasses a kit for use in the therapeutic treatment of various disease states, such as cancer, AIDS or influenza, said kit comprising a pharmaceutical composition comprising insect cells containing baculovirus. said baculovirus containing an isolated nucleic acid segment encoding a selected non-surface expressed therapeutic protein or peptide.